Genus Mucuna belongs to the family Fabaceae is one of the potential underutilized legumes which include 150 species of annual and perennial species of pantropical distribution. Members of this genus possess several promising nutritional and agronomic attributes it has received wide-ranging attention from pharmaceutical industries, nutritional chemists and agronomists alike in recent years. The evolution and relationship among different Mucuna taxa (both at species and sub-species level) of India have remained unknown except M. pruriens. Because of this, it is necessary to conduct research at the species level as well as to assess phenetic relationships among different taxa in India to place the genus in right taxonomic and phylogenetic perspective. The Consortium for the Barcode of Life (CBOL) – Plant working group revealed the utility of nuclear Internal Transcribed Spacer (nrITS) region of the nuclear ribosomal cistron (18S-5.8S-26S) and chloroplast regions like trnHpsbA, matK and rbcL towards developing the plant DNA barcodes. Though these regions are considered as universal barcode regions, it observed from literature that to get successful amplification of these regions many PCR parameters needs to be optimized. Here we report different amplification parameters optimized for the amplification of nrITS2, trnH-psbA, matK and rbcL regions in Mucuna species of India collected from different geographical locations. The amplified regions from the optimized protocols were sequenced and obtained sequences after editing were queried in NCBI BLAST to confirm that they distinctly match the respective barcode regions of the Mucuna species. The results of these analysis revealed that obtained sequences were authentic and could be used for phylogenetic studies and DNA barcoding of Mucuna species of India.